Follistatin domain containing proteins

ABSTRACT

The present invention relates to the use of proteins comprising at least one follistatin domain to modulate the level or activity of growth and differentiation factor-8 (GDF-8). More particularly, the invention relates to the use of proteins comprising at least one follistatin domain, excluding follistatin itself, for treating disorders that are related to modulation of the level or activity of GDF-8. The invention is useful for treating muscular diseases and disorders, particularly those in which an increase in muscle tissue would be therapeutically beneficial. The invention is also useful for treating diseases and disorders related to metabolism, adipose tissue, and bone degeneration.

This application claims the benefit of U.S. Provisional Application No.60/357,846, filed Feb. 21, 2002, and U.S. Provisional Application No.60/434,645, filed Dec. 20, 2002.

FIELD OF THE INVENTION

The present invention relates to the use of proteins comprising at leastone follistatin domain to modulate the level or activity of growth anddifferentiation factor-8 (GDF-8). More particularly, the inventionrelates to the use of proteins comprising at least one follistatindomain, excluding follistatin itself, for treating disorders that arerelated to modulation of the level or activity of GDF-8. The inventionis useful for treating muscular diseases and disorders, particularlythose in which an increase in muscle tissue would be therapeuticallybeneficial. The invention is also useful for treating diseases anddisorders related to metabolism, adipose tissue, and bone degeneration.

BACKGROUND OF THE INVENTION

Growth and differentiation factor-8 (GDF-8), also known as myostatin, isa member of the transforming growth factor-beta (TGF-β) superfamily ofstructurally related growth factors, all of which possess importantphysiological growth-regulatory and morphogenetic properties (Kingsleyet al. (1994) Genes Dev., 8: 133-46; Hoodless et al. (1998) Curr. TopicsMicrobiol. Immunol., 228: 235-72). GDF-8 is a negative regulator ofskeletal muscle mass, and there is considerable interest in identifyingfactors which regulate its biological activity. For example, GDF-8 ishighly expressed in the developing and adult skeletal muscle. The GDF-8null mutation in transgenic mice is characterized by a markedhypertrophy and hyperplasia of the skeletal muscle (McPherron et al.(1997) Nature, 387: 83-90). Similar increases in skeletal muscle massare evident in naturally occurring mutations of GDF-8 in cattle (Ashmoreet al. (1974) Growth, 38: 501-507; Swatland and Kieffer (1994) J. Anim.Sci., 38: 752-757; McPherron and Lee (1997) Proc. Nat. Acad. Sci.U.S.A., 94: 12457-12461; and Kambadur et al. (1997) Genome Res., 7:910-915). Recent studies have also shown that muscle wasting associatedwith HIV-infection in humans is accompanied by increases in GDF-8protein expression (Gonzalez-Cadavid et al. (1998) Proc. Natl. Acad Sci.U.S.A., 95: 14938-43). In addition, GDF-8 can modulate the production ofmuscle-specific enzymes (e.g., creatine kinase) and modulate myoblastcell proliferation (WO 00/43781).

A number of human and animal disorders are associated with loss of orfunctionally impaired muscle tissue. To date, very few reliable oreffective therapies exist for these disorders. However, the terriblesymptoms associated with these disorders may be substantially reduced byemploying therapies that increase the amount of muscle tissue inpatients suffering from the disorders. While not curing the conditions,such therapies would significantly improve the quality of life for thesepatients and could ameliorate some of the effects of these diseases.Thus, there is a need in the art to identify new therapies that maycontribute to an overall increase in muscle tissue in patients sufferingfrom these disorders.

In addition to its growth-regulatory and morphogenetic properties inskeletal muscle, GDF-8 may also be involved in a number of otherphysiological processes (e.g., glucose homeostasis), as well as abnormalconditions, such as in the development of type 2 diabetes and adiposetissue disorders, such as obesity. For example, GDF-8 modulatespreadipocyte differentiation to adipocytes (Kim et al. (2001) B.B.R.C.281: 902-906). Thus, modulation of GDF-8 may be useful for treatingthese diseases, as well.

The GDF-8 protein is synthesized as a precursor protein consisting of anamino-terminal propeptide and a carboxy-terminal mature domain(McPherron and Lee, (1997) Proc. Nat. Acad. Sci. U.S.A., 94:12457-12461). Before cleavage, the precursor GDF-8 protein forms ahomodimer. The amino-terminal propeptide is then cleaved from the maturedomain. The cleaved propeptide may remain noncovalently bound to themature domain dimer, inactivating its biological activity (Miyazono etal. (1988) J. Biol. Chem., 263: 6407-6415; Wakefield et al. (1988) J.Biol. Chem., 263: 7646-7654; and Brown et al. (1990) Growth Factors, 3:35-43). It is believed that two GDF-8 propeptides bind to the GDF-8mature dimer (Thies et al. (2001) Growth Factors, 18: 251-259). Due tothis inactivating property, the propeptide is known as the“latency-associated peptide” (LAP), and the complex of mature domain andpropeptide is commonly referred to as the “small latent complex” (Gentryand Nash (1990) Biochemistry, 29:6851-6857; Derynck et al. (1995)Nature, 316:701-705; and Massague (1990) Ann. Rev. Cell Biol., 12:597-641). Other proteins are also known to bind to GDF-8 or structurallyrelated proteins and inhibit their biological activity. Such inhibitoryproteins include follistatin (Gamer et al. (1999) Dev. Biol., 208:222-232). The mature domain of GDF-8 is believed to be active as ahomodimer when the propeptide is removed.

Clearly, GDF-8 is involved in the regulation of many critical biologicalprocesses. Due to its key function in these processes, GDF-8 may be adesirable target for therapeutic intervention. In particular,therapeutic agents that inhibit the activity of GDF-8 may be used totreat human or animal disorders in which an increase in muscle tissuewould be therapeutically beneficial.

Known proteins comprising at least one follistatin domain play roles inmany biological processes, particularly in the regulation of TGF-βsuperfamily signaling and the regulation of extracellularmatrix-mediated processes such as cell adhesion. Follistatin,follistatin related gene (FLRG, FSRP), and follistatin-related protein(FRP) have all been linked to TGF-β signaling, either throughtranscriptional regulation by TGF-β (Bartholin et al. (2001) Oncogene,20: 5409-5419; Shibanuma et al. (1993) Eur. J. Biochem. 217: 13-19) orby their ability to antagonize TGF-β signaling pathways (Phillips and deKretser (1998) Front. Neuroendocrin., 19: 287-322; Tsuchida et al.(2000) J. Biol. Chem., 275: 40788-40796; Patel et al. (1996) Dev. Biol.,178: 327-342; Amthor et al. (1996) Dev. Biol., 178: 343-362). Proteinnames in parentheses are alternative names.

Insulin growth factor binding protein 7 (IGFBP7, mac25), which compriseat least one follistatin domain, binds to insulin and blocks subsequentinteraction with the insulin receptor. In addition, IGFBP7 has beenshown to bind to activin, a TGF-β family member (Kato (2000) Mol. Med.,6:126-135).

Agrins and agrin related proteins contain upwards of nine follistatindomains and are secreted from nerve cells to promote the aggregation ofacetylcholine receptors and other molecules involved in the formation ofsynapses. It has been suggested that the follistatin domains may serveto localize growth factors to the synapse (Patthy et al. (1993) TrendsNeurosci., 16: 76-81).

Osteonectin (SPARC, BM40) and hevin (SC1, mast9, QR1) are closelyrelated proteins that interact with extracellular matrix proteins andregulate cell growth and adhesion (Motamed (1999) Int. J. Biochem. Cell.Biol., 31: 1363-1366; Girard and Springer (1996) J. Biol. Chem., 271:4511-4517). These proteins comprise at least one follistatin domain.

Other follistatin domain proteins have been described or uncovered fromthe NCBI database (National Center for Biotechnology Information,Bethesda, Md., USA), however their functions are presently unknown.These proteins include U19878 (G01639, very similar to tomoregulin-1),T46914, human GASP1 (GDF-associated serum protein 1; described herein;FIG. 7), human GASP2 (WFIKKN; Trexler et al. (2001) Proc. Natl. Acad.Sci. U.S.A., 98: 3705-3709; FIG. 9), and the proteoglycan family oftestican (SPOCK) proteins (Alliel et al.(1993) Eur. J. Biochem., 214:347-350). Amino acid and nucleotide sequences for mouse GASP1 (FIG. 6)and mouse GASP2 (FIG. 8) were also determined from the Celera database(Rockville, Md.). As described herein, the nucleotide sequence of clonedmouse GASP1 matched the predicted Celera sequence, with the exception ofsome base substitutions in wobble codons that did not change thepredicted amino acid sequence (see FIG. 13).

SUMMARY OF THE INVENTION

Accordingly, the invention relates to proteins, other than follistatin,comprising a unique structural feature, namely, the presence of at leastone follistatin domain. Follistatin itself is not encompassed by theinvention. The proteins comprising at least one follistatin domain arespecifically reactive with a mature GDF-8 protein or a fragment thereof,whether the GDF-8 protein is in monomeric form, a dimeric active form,or complexed in the GDF-8 latent complex. Proteins comprising at leastone follistatin domain may bind to an epitope on the mature GDF-8protein that results in a reduction in one or more of the biologicalactivities associated with GDF-8, relative to a mature GDF-8 proteinthat is not bound by the same protein.

The present invention provides methods for modulating the effects ofGDF-8 on cells. Such methods comprise administering an effective amountof a protein comprising at least one follistatin domain. The presentinvention also encompasses methods for expressing a protein in a cell byadministering a DNA molecule encoding a protein comprising at least onefollistatin domain.

According to the invention, proteins comprising at least one follistatindomain may be administered to a patient, in a therapeutically effectivedose, to treat or prevent medical conditions in which an increase inmuscle tissue would be therapeutically beneficial. Embodiments includetreatment of diseases, disorders, and injuries involving cells andtissue that are associated with the production, metabolism, or activityof GDF-8.

Proteins comprising at least one follistatin domain may be prepared in apharmaceutical preparation. The pharmaceutical preparation may containother components that aid in the binding of the mature GDF-8 protein orfragments thereof, whether it is in monomeric form, dimeric active form,or complexed in the GDF-8 latent complex.

In addition, proteins comprising at least one follistatin domain may beused as a diagnostic tool to quantitatively or qualitatively detectmature GDF-8 protein or fragments thereof, whether it is in monomericform, dimeric active form, or complexed in the GDF-8 latent complex. Forexample, proteins comprising at least one follistatin domain may be usedto detect the presence, absence, or amount of GDF-8 protein in a cell,bodily fluid, tissue, or organism. The presence or amount of matureGDF-8 protein detected may be correlated with one or more of the medicalconditions listed herein.

Proteins comprising at least one follistatin domain may be provided in adiagnostic kit to detect mature GDF-8 protein or fragments thereof,whether it is in monomeric form, dimeric active form, or complexed inthe GDF-8 latent complex, and help correlate the results with one ormore of the medical conditions described herein. Such a kit may compriseat least one protein comprising at least one follistatin domain, whetherit is labeled or unlabeled, and at least one agent that bind to thisproteins, such as a labeled antibody. The kit may also include theappropriate biological standards and control samples to which one couldcompare the results of the experimental detection. It may also includebuffers or washing solutions and instructions for using the kit.Structural components may be included on which one may carry out theexperiment, such as sticks, beads, papers, columns, vials, or gels.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows antibody purification of the GDF-8 complex from wild-typemouse serum. A silver stained reducing gel shows proteins purified fromwild type mouse serum using the JA16 monoclonal antibody covalentlycoupled to agarose beads. A control purification (0) with mock-coupledbeads was performed in parallel. Subsequent elutions with buffer (mockelute), a competing peptide, and SDS sample buffer revealed two visibleprotein bands which were specifically eluted with peptide from theJA16-conjugated beads (indicated by arrows).

FIG. 2 shows the identification of mature and unprocessed GDF-8 inaffinity purified samples from normal mouse serum. FIG. 2A shows arepresentative MS/MS spectrum of a GDF-8 derived peptide (SEQ ID NO:19)identified from the 12 kDa band visible in the affinity purified sample.Both N-terminal fragment ions (b ions) and C-terminal fragment ions (yions) are visible. Notably, the most intense y fragment ions result fromfragmentation before the proline residue, a common characteristic ofproline containing peptides. FIG. 2B shows a western blot probed with apolyclonal antibody that recognizes the mature region of GDF-8,confirming the presence of GDF-8 in the affinity purified samples. Boththe mature and unprocessed forms of GDF-8 are visible.

FIG. 3 shows the GDF-8 propeptide and follistatin-like related gene(FLRG) bind to circulating GDF-8 isolated from normal mouse serum.Representative MS/MS spectra from GDF-8 propeptide (SEQ ID NO:23) (FIG.3A) and FLRG (SEQ ID NO:30) (FIG. 3C) derived peptides identified in the36 kDa band are shown. FIG. 3B shows a western blot of affinity purifiedGDF-8 complex probed with a polyclonal antibody that specificallyrecognizes the propeptide region of GDF-8, confirming the massspectrometric identification of this protein in the GDF-8 complex. Boththe clipped propeptide and unprocessed GDF-8 are visible—at longerexposures, unprocessed GDF-8 can also be seen in the SDS eluted sample.FIG. 3D shows a western blot of affinity purified GDF-8 complex probedwith a monoclonal antibody to FLRG.

FIG. 4 shows results from a thorough analysis of a large scale GDF-8purification that identified GDF-8 propeptide, FLRG, and a novel proteinas the major GDF-8 binding proteins in serum. A silver stained gel wasdissected into 13 slices from the peptide eluted sample of both negativecontrol and JA16 immunoprecipitates. The proteins in each slice weredigested with trypsin and identified using nanoflow-LC-MS/MS anddatabase searching. Proteins unique to the JA16 sample included onlyunprocessed and mature GDF-8, GDF-8 propeptide, FLRG, and a novelmultidomain protease inhibitor (GDF-associated serum protein 1, GASP1).These proteins were identified from the noted regions of the gel.

FIG. 5 shows that a novel multidomain protease inhibitor, GASP1, isbound to GDF-8 in serum. FIGS. 5A (peptide assigned SEQ ID NO:31) and 5B(peptide assigned SEQ ID NO:33) show representative MS/MS spectra fromtwo GASP1 peptides, identified in band 3 of the silver stained gel ofFIG. 4.

FIG. 6A shows the predicted nucleotide sequence to mouse GASP1.

FIG. 6B shows a predicted alternative nucleotide sequence to mouseGASP1. FIG. 6C shows the predicted amino acid sequence encoded by thenucleotide sequences shown in FIGS. 6A and 6B. The protein sequencesencoded by the two nucleotide sequences are identical because thenucleotide differences are all in wobble codon positions. Thefollistatin domain is shown in bold and underlined.

FIG. 7A shows the predicted nucleotide sequence of human GASP1. FIG. 7Bshows the corresponding predicted amino acid sequence. The follistatindomain is shown in bold and underlined. FIG. 7C shows the predictednucleotide sequence of human GASP1 using an alternative start site. FIG.7D shows the corresponding predicted amino acid sequence. Thefollistatin domain is shown in bold and underlined. The end of thesequence is denoted by the asterisk.

FIG. 8A shows the predicted nucleotide sequence to mouse GASP2, whileFIG. 8B shows the corresponding predicted amino acid sequence. Thefollistatin domain is shown in bold and underlined.

FIG. 9A shows the predicted nucleotide sequence to human GASP2, whileFIG. 9B shows the corresponding predicted amino acid sequence. Thefollistatin domain is shown in bold and underlined.

FIG. 10 shows that mouse GASP1 is expressed in many adult tissues andduring development. The figure shows tissue expression profiles of mouseGASP1. A 551 bp fragment of GASP1 was amplified from normalizedfirst-strand cDNA panels from Clontech (Palo Alto, Calif.). A portion ofglyceraldehyde-3-phosphate dehydrogenase (G3PDH) was amplified as acontrol. G3PDH expression is known to be high in skeletal muscle and lowin testis. The cDNA panels were normalized against β-actin,phospholipase A2, and ribosomal protein S29, in addition to G3PDH.

FIG. 11A shows proteins isolated from human serum. Proteins from a JA16immunoprecipitate or a control sample (0) were eluted in a mock PBSelution, a competing peptide elution, or a SDS elution. The proteins inthe indicated regions of the gel were digested with trypsin and analyzedby LS-MS/MS and database searching. The proteins present in the JA16sample but not in the control sample were mature GDF-8 (band 16), GDF-8propeptide and FLRG (band 11), and human GASP1 (band 4). FIG. 11B showsa western blot of an identical JA16 immunoprecipitate probed with anantibody that recognizes mature GDF-8. Bands corresponding to mature andunprocessed GDF-8 isolated from human serum are visible.

FIG. 12 shows representative mass spectra of a peptide derived fromGDF-8 and associated proteins isolated from bands 4, 11, and 16 (FIG.11). The peptide sequence and N-terminal (b ions) and C-terminal (yions) are shown. A complete listing of identified peptides is providedin Table 1. Spectra are shown from a GASP1 peptide (SEQ ID NO:44) (FIG.12A), a FLRG peptide (SEQ ID NO:41) (FIG. 12B), a GDF-8 propeptidepeptide (SEQ ID NO:24) (FIG. 12C), and a mature GDF-8 peptide (SEQ IDNO:13) (FIG. 12D).

FIG. 13 shows the nucleotide (SEQ ID NO:48) and amino acid (SEQ IDNO:49) sequences of cloned mouse GASP1. The peptides identified by massspectrometry in JA16 affinity-purified samples are underlined. The endof the sequence is denoted by the asterisk.

FIG. 14A shows the domain structure of GASP1. GASP1 has a signalsequence/cleavage site after amino acid 29. In addition, GASP1 containstwo Kunitz/BPTI serine protease inhibitor domains, a follistatin domain(including a Kazal serine protease inhibitor motif) and a netrin domain,which may inhibit metalloproteases. FIG. 14B shows the phylogenetic treeof GASP1 and GASP2 predicted from the mouse and human genomic sequences.Mouse and human GASP1 are 90% identical. GASP1 and GASP2 are 54%identical.

FIG. 15 shows that recombinantly-produced GASP1 binds separately to bothGDF-8 and GDF-8 propeptide. (A) JA16 was used to immunoprecipitate GDF-8from mock- or GASP1-V5-His transfected COS cell conditioned mediasupplemented with recombinant purified GDF-8 and/or propeptide. Westernblots with anti-V5 (top panel), anti-GDF-8 (middle panel), oranti-propeptide polyclonal antibodies were used to determine whetherthese proteins were present in the immunoprecipitate. (B)Recombinantly-produced GASP1 protein was immunoprecipitated by anti-V5tag antibodies from mock- or GASP1-V5-His conditioned media supplementedwith recombinant purified GDF-8 and/or propeptide. The immunoprecipitatewas analyzed by western blotting as in (A).

FIG. 16 shows that GASP1 inhibits the biological activity of GDF-8 andthe highly related BMP-11, but not activin or TGF-β. Various dilutionsof conditioned media from mock (open circles) or GASP1-V5-His (filledsquares) transfectants were incubated with (A) 10 ng/ml GDF-8, (B) 10ng/ml BMP-11, (C) 10 ng/ml activin, or (D) 0.5 ng/ml TGF-β. Thesesamples were then subjected to a luciferase reporter activity assay inA204 (A-C) or RD (D) cells to determine the activity of the added growthfactors. Luciferase activity is shown in relative luciferase units. Theactivity resulting from each of the growth factors alone is shown by thefilled diamonds and short dashed line. Without addition of any growthfactor, the background activity in the assay is low, as shown by thelong dashed line with no symbols.

FIG. 17 shows the potency of GASP1 inhibition of GDF-8. Purified GASP1was tested for its ability to inhibit 20 ng/ml of myostatin in the(CAGA)₁₂ (SEQ ID NO:53) luciferase reporter assay in RD cells (filledsquares). The activity resulting from GDF-8 alone is shown by the filleddiamonds and short dashed line. The activity present when no growthfactors are added is shown by the long dashed line.

DEFINITIONS

The term “follistatin domain” refers to an amino acid domain or anucleotide domain encoding for an amino acid domain, characterized bycysteine rich repeats. A follistatin domain typically encompasses a65-90 amino acid span and contains 10 conserved cysteine residues and aregion similar to Kazal serine protease inhibitor domains. In general,the loop regions between the cysteine residues exhibit sequencevariability in follistatin domains, but some conservation is evident.The loop between the fourth and fifth cysteines is usually small,containing only 1 or 2 amino acids. The amino acids in the loop betweenthe seventh and eighth cysteines are generally the most highly conservedcontaining a consensus sequence of (G,A)-(S,N)(S,N,T)-(D,N)-(G,N)followed by a (T,S)-Y motif. The region between the ninth and tenthcysteines generally contains a motif containing two hydrophobic residues(specifically V, I, or L) separated by another amino acid.

The term “protein comprising at least one follistatin domain” refers toproteins comprising at least one, but possibly more than one follistatindomain. The term also refers to any variants of such proteins (includingfragments; proteins with substitution, addition or deletion mutations;and fusion proteins) that maintain the known biological activitiesassociated with the native proteins, especially those pertaining toGDF-8 binding activity, including sequences that have been modified withconservative or non-conservative changes to the amino acid sequence.These proteins may be derived from any source, natural or synthetic. Theprotein may be human or derived from animal sources, including bovine,chicken, murine, rat, porcine, ovine, turkey, baboon, and fish.Follistatin itself is not encompassed by the invention.

The terms “GDF-8” or “GDF-8 protein” refer to a specific growth anddifferentiation factor. The terms include the full length unprocessedprecursor form of the protein, as well as the mature and propeptideforms resulting from post-translational cleavage. The terms also referto any fragments of GDF-8 that maintain the known biological activitiesassociated with the protein, including sequences that have been modifiedwith conservative or non-conservative changes to the amino acidsequence. These GDF-8 molecules may be derived from any source, naturalor synthetic. The protein may be human or derived from animal sources,including bovine, chicken, murine, rat, porcine, ovine, turkey, baboon,and fish. Various GDF-8 molecules have been described in McPherron etal. (1997) Proc. Natl. Acad. Sci. USA, 94: 12457-12461.

“Mature GDF-8” refers to the protein that is cleaved from thecarboxy-terminal domain of the GDF-8 precursor protein. The mature GDF-8may be present as a monomer, homodimer, or in a GDF-8 latent complex.Depending on the in vivo or in vitro conditions, mature GDF-8 mayestablish an equilibrium between any or all of these different forms. Itis believed to be biologically active as homodimer. In its biologicallyactive form, the mature GDF-8 is also referred to as “active GDF-8.”

“GDF-8 propeptide” refers to the polypeptide that is cleaved from theamino-terminal domain of the GDF-8 precursor protein. The GDF-8propeptide is capable of binding to the propeptide binding domain on themature GDF-8.

“GDF-8 latent complex” refers to the complex of proteins formed betweenthe mature GDF-8 homodimer and the GDF-8 propeptide. It is believed thattwo GDF-8 propeptides associate with the two molecules of mature GDF-8in the homodimer to form an inactive tetrameric complex. The latentcomplex may include other GDF inhibitors in place of or in addition toone or more of the GDF-8 propeptides.

The phrase “GDF-8 activity” refers to one or more of physiologicallygrowth-regulatory or morphogenetic activities associated with activeGDF-8 protein. For example, active GDF-8 is a negative regulator ofskeletal muscle. Active GDF-8 can also modulate the production ofmuscle-specific enzymes (e.g., creatine kinase), stimulate myoblast cellproliferation, and modulate preadipocyte differentiation to adipocytes.GDF-8 is also believed to increase sensitivity to insulin and glucoseuptake in peripheral tissues, particularly in skeletal muscle oradipocytes. Accordingly, GDF-8 biological activities include but are notlimited to inhibition of muscle formation, inhibition of muscle cellgrowth, inhibition of muscle development, decrease in muscle mass,regulation of muscle-specific enzymes, inhibition of myoblast cellproliferation, modulation of preadipocyte differentiation to adipocytes,increasing sensitivity to insulin, regulations of glucose uptake,glucose hemostasis, and modulate neuronal cell development andmaintenance.

The terms “isolated” or “purified” refer to a molecule that issubstantially free of its natural environment. For instance, an isolatedprotein is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which it isderived. The phrase “substantially free of cellular material” refers topreparations where the isolated protein is at least 70% to 80% (w/w)pure, at least 80%89% (w/w) pure, at least 90-95% pure, or at least 96%,97%, 98%, 99% or 100% (w/w) pure.

The term “LC-MS/MS” refers to liquid chromatography in line with a massspectrometer programmed to isolate a molecular ion of particularmass/charge ratio, fragment this ion, and record the mass/charge ratioof the fragment ions. When analyzing peptide samples this techniqueallows upstream separation of complex samples through liquidchromatography, followed by the recording of fragment ion masses andsubsequent determination of the peptide sequence.

The term “MS/MS” refers to the process of using a mass spectrometer toisolate a molecular ion of a particular mass/charge ratio, fragment thision, and record the mass/charge ratio of the resulting fragment ions.The fragment ions provide information about the sequence of a peptide.

The term “treating” or “treatment” refers to both therapeutic treatmentand prophylactic or preventative measures. Those in need of treatmentmay include individuals already having a particular medical disorder aswell as those who may ultimately acquire the disorder (i.e., thoseneeding preventative measures). The term treatment includes bothmeasures that address the underlying cause of a disorder and measuresthat reduce symptoms of a medical disorder without necessarily affectingits cause. Thus, improvement of quality of life and amelioration ofsymptoms are considered treatment, as are measures that counteract thecause of a disorder.

The term “medical disorder” refers to disorders of muscle, bone, orglucose homeostasis, and include disorders associated with GDF-8 and/orother members of the TGF-.beta. superfamily (e.g., BMP-1 1). Examples ofsuch disorders include, but are not limited to, metabolic diseases anddisorders such as insulin-dependent (type 1) diabetes mellitus,noninsulin-dependent (type 2) diabetes mellitus, hyperglycemia, impairedglucose tolerance, metabolic syndrome (e.g., syndrome X), and insulinresistance induced by trauma (e.g., burns or nitrogen imbalance), andadipose tissue disorders (e.g., obesity); muscle and neuromusculardisorders such as muscular dystrophy (including but not limited tosevere or benign X-linked muscular dystrophy, limb-girdle dystrophy,facioscapulohumeral dystrophy, myotonic dystrophy, distal musculardystrophy, progressive dystrophic ophthalmoplegia, oculopharyngealdystrophy, Duchenne's muscular dystrophy, and Fakuyama-type congenitalmuscular dystophy); amyotrophic lateral sclerosis (ALS); muscle atrophy;organ atrophy; frailty; carpal tunnel syndrome; congestive obstructivepulmonary disease; congenital myopathy; myotonia congenital; familialperiodic paralysis; paroxysmal myoglobinuria; myasthenia gravis;Eaton-Lambert syndrome; secondary myasthenia; denervation atrophy;paroxymal muscle atrophy; and sarcopenia, cachexia and other musclewasting syndromes. Other examples include osteoporosis, especially inthe elderly and/or postmenopausal women; glucocorticoid-inducedosteoporosis; osteopenia; osteoarthritis; osteoporosis-relatedfractures; and traumatic or chronic injury to muscle tissue. Yet furtherexamples include low bone mass due to chronic glucocorticoid therapy,premature gonadal failure, androgen suppression, vitamin D deficiency,secondary hyperparathyroidism, nutritional deficiencies, and anorexianervosa.

The term “increase in mass” refers to the presence of a greater amountof muscle after treatment with proteins comprising at least onefollistatin domain relative to the amount of muscle mass present beforethe treatment.

The term “therapeutic benefit” refers to an improvement of symptoms of adisorder, a slowing of the progression of a disorder, or a cessation inthe progression of a disorder. The therapeutic benefit is determined bycomparing an aspect of a disorder, such as the amount of muscle mass,before and after at least one protein comprising at last one follistatindomain is administered.

The term “modulating” refers to varying a property of a protein byincreasing, decreasing, or inhibiting the activity, behavior, or amountof the protein. For example, proteins comprising at least onefollistatin domain may modulate GDF-8 by inhibiting its activity.

The term “stabilizing modification” is any modification known in the artor set forth herein capable of stabilizing a protein, enhancing the invitro half life of a protein, enhancing circulatory half life of aprotein and/or reducing proteolytic degradation of a protein. Suchstabilizing modifications include but are not limited to fusion proteins(including, for example, fusion proteins comprising a protein comprisingat least one follistatin domain and a second protein), modification of aglycosylation site (including, for example, addition of a glycosylationsite to a protein comprising at least one follistatin domain), andmodification of carbohydrate moiety (including, for example, removal ofcarbohydrate moieties from a protein comprising at least one follistatindomain). In the case of a stabilizing modification which comprises afusion protein (e.g., such that a second protein is fused to a proteincomprising at least one follistatin domain), the second protein may bereferred to as a “stabilizer portion” or “stabilizer protein.” Forexample, a protein a human protein comprising at least one follistatindomain may be fused with an IgG molecule, wherein IgG acts as thestabilizer protein or stabilizer portion. As used herein, in addition toreferring to a second protein of a fusion protein, a “stabilizerportion” also includes nonproteinaceous modifications such as acarbohydrate moiety, or nonproteinaceous polymer.

The term “Fc region of an IgG molecule” refers to the Fc domain of animmunoglobulin of the isotype IgG, as is well known to those skilled inthe art. The Fc region of an IgG molecule is that portion of IgGmolecule (IgG1, IgG2, IgG3, and IgG4) that is responsible for increasingthe in vivo serum half-life of the IgG molecule.

“In vitro half life” refers to the stability of a protein measuredoutside the context of a living organism. Assays to measure in vitrohalf life are well known in the art and include but are not limited toSDS-PAGE, ELISA, cell-based assays, pulse-chase, western blotting,northern blotting, etc. These and other useful assays are well known inthe art.

“In vivo half life” refers to the stability of a protein in an organism.In vivo half life may be measured by a number of methods known in theart including but not limited to in vivo serum half life, circulatoryhalf life, and assays set forth in the examples herein.

“In vivo serum half life” refers to the half-life of a proteincirculating in the blood of an organism. Methods known in the art may beused to measure in vivo serum half life. For example, radioactiveprotein can be administered to an animal and the amount of labeledprotein in the serum can be monitored over time.

To assist in the identification of the sequences listed in thespecification and figures, the following table is provided, which liststhe SEQ ID NO, the figure location, and a description of the sequence.

SEQ ID NO: REFERENCE DESCRIPTION  1 FIG. 6A predicted mouse GASP1nucleotide sequence  2 FIG. 6B predicted mouse GASP1 alternativenucleotide sequence  3 FIG. 6C predicted mouse GASP1 amino acid sequenceencoded by both SEQ ID NOS:1 and 2  4 FIG. 7A predicted human GASP1nucleotide sequence  5 FIG. 7B predicted human GASP1 amino acid sequenceencoded by SEQ ID NO:4  6 FIG. 7C predicted human GASP1 nucleotidesequence, alternative start site  7 FIG. 7D predicted human GASP1 aminoacid sequence, alternative start site encoded by SEQ ID NO:6  8 FIG. 8Apredicted mouse GASP2 nucleotide sequence  9 FIG. 8B predicted mouseGASP2 amino acid sequence encoded by SEQ ID NO:8 10 FIG. 9A predictedhuman GASP2 nucleotide sequence 11 FIG. 9B predicted human GASP2 aminoacid sequence encoded by SEQ ID NO:10 12 Example 2 competing peptide13-20 Table 1, mouse GDF-8 peptides Examples 5, 6 21-27 Table 1, mouseGDF-8 propeptide peptides Examples 5, 6 28-30 Table 1, mouse FLRGpeptides Example 5 31-35 Table 1, mouse GASP1 peptides Examples 5, 736-37 Table 1, human GDF-8 peptides Example 8 38-39 Table 1, human GDF-8propeptide peptides Example 8 40-42 Table 1, human FLRG peptides Example8 43-45 Table 1, human GASP1 peptides Example 8 46 Example 7 forwardprimer 47 Example 7 reverse primer 48 FIG. 13 cloned mouse GASP1nucleotide sequence 49 FIG. 13 cloned mouse GASP1 amino acid sequenceencoded by SEQ ID NO:48 50 Example 9 forward primer 51 Example 9 reverseprimer 52 Example 9 illustrative N-terminal peptide sequence 53 Example11 synthetic oligonucleotide

DETAILED DESCRIPTION OF THE INVENTION

Proteins Comprising at Least One Follistatin Domain

The present invention relates to proteins, other than follistatin,having a unique structural feature, namely, that they comprise at leastone follistatin domain. Follistatin itself is not encompassed by theinvention. It is believed that proteins containing at least onefollistatin domain will bind and inhibit GDF-8. Examples of proteinshaving at least one follistatin domain include, but are not limited tofollistatin-like related gene (FLRG), FRP (flik, tsc 36), agrins,osteonectin (SPARC, BM40), hevin (SC1, mast9, QR1), IGFBP7 (mac25), andU19878. GASP1, comprising the nucleotide and amino acid sequencesprovided in FIGS. 6 and 7, and GASP2, comprising the nucleotide andamino acid sequences provided in FIGS. 8 and 9, are other examples ofproteins comprising at least one follistatin domain.

A follistatin domain, as stated above, is defined as an amino aciddomain or a nucleotide domain encoding for an amino acid domain,characterized by cysteine rich repeats. A follistatin domain typicallyencompasses a 65-90 amino acid span and contains 10 conserved cysteineresidues and a region similar to Kazal serine protease inhibitordomains. In general, the loop regions between the cysteine residuesexhibit sequence variability in follistatin domains, but someconservation is evident. The loop between the fourth and fifth cysteinesis usually small, containing only 1 or 2 amino acids. The amino acids inthe loop between the seventh and eighth cysteines are generally the mosthighly conserved containing a consensus sequence of(G,A)-(S,N)-(S,N,T)-(D,N)-(G,N) followed by a (T,S)-Y motif. The regionbetween the ninth and tenth cysteines generally contains a motifcontaining two hydrophobic residues (specifically V, I, or L) separatedby another amino acid.

Proteins comprising at least one follistatin domain, which may bindGDF-8, may be isolated using a variety of methods. For example, one mayuse affinity purification using GDF-8, as exemplified in the presentinvention. In addition, one may use a low stringency screening of a cDNAlibrary, or use degenerate PCR techniques using a probe directed towarda follistatin domain. As more genomic data becomes available, similaritysearching using a number of sequence profiling and analysis programs,such as MotifSearch (Genetics Computer Group, Madison, Wis.),ProfileSearch (GCG), and BLAST (NCBI) could be used to find novelproteins containing significant homology with known follistatin domains.

One of skill in the art will recognize that both GDF-8 or proteinscomprising at least one follistatin domain may contain any number ofconservative changes to their respective amino acid sequences withoutaltering their biological properties. Such conservative amino acidmodifications are based on the relative similarity of the amino acidside-chain substituents, for example, their hydrophobicity,hydrophilicity, charge, size, and the like. Exemplary conservativesubstitutions which take various of the foregoing characteristics intoconsideration are well known to those of skill in the art and include:arginine and lysine; glutamate and aspartate; serine and threonine;glutamine and asparagine; and valine, leucine, and isoleucine.Furthermore, proteins comprising at least one follistatin domain may beused to generate functional fragments comprising at least onefollistatin domain. It is expected that such fragments would bind andinhibit GDF-8. In an embodiment of the invention, proteins comprising atleast one follistatin domain specifically bind to mature GDF-8 or afragment thereof, whether it is in monomeric form, active dimer form, orcomplexed in a GDF-8 latent complex, with an affinity of between 0.001and 100 nM, or between 0.01 and 10 nM, or between 0.1 and 1 nM.

Nucleotide and Protein Sequences

While not always necessary, if desired, one of ordinary skill in the artmay determine the amino acid or nucleic acid sequences of a novelproteins comprising at least one follistatin domain. For example, thepresent invention provides the amino acid and nucleotide sequences forGASP1 and GASP2, as shown in FIGS. 6-9.

The present invention also include variants, homologues, and fragmentsof such nucleic and amino acid sequences. For example, the nucleic oramino acid sequence may comprise a sequence at least 70% to 79%identical to the nucleic or amino acid sequence of the native protein,or at least 80% to 89% identical, or at least 90% to 95% identical, orat least 96% to 100% identical. One of skill in the art will recognizethat the region that binds GDF-8 can tolerate less sequence variationthan the other portions of the protein not involved in binding. Thus,these non-binding regions of the protein may contain substantialvariations without significantly altering the binding properties of theprotein. However, one of skill in the art will also recognize that manychanges can be made to specifically increase the affinity of the proteinfor its target. Such affinity-increasing changes are typicallydetermined empirically by altering the protein, which may be in thebinding region, and testing the ability to bind GDF-8 or the strength ofthe binding. All such alterations, whether within or outside the bindingregion, are included in the scope of the present invention.

Relative sequence similarity or identity may be determined using the“Best Fit” or “Gap” programs of the Sequence Analysis Software Package™(Version 10; Genetics Computer Group, Inc., University of WisconsinBiotechnology Center, Madison, Wis.). “Gap” utilizes the algorithm ofNeedleman and Wunsch (Needleman and Wunsch, 1970) to find the alignmentof two sequences that maximizes the number of matches and minimizes thenumber of gaps. “BestFit” performs an optimal alignment of the bestsegment of similarity between two sequences. Optimal alignments arefound by inserting gaps to maximize the number of matches using thelocal homology algorithm of Smith and Waterman (Smith and Waterman,1981; Smith, et al., 1983).

The Sequence Analysis Software Package described above contains a numberof other useful sequence analysis tools for identifying homologues ofthe presently disclosed nucleotide and amino acid sequences. Forexample, the “BLAST” program (Altschul, et al., 1990) searches forsequences similar to a query sequence (either peptide or nucleic acid)in a specified database (e.g., sequence databases maintained at theNCBI; “FastA” (Lipman and Pearson, 1985; see also Pearson and Lipman,1988; Pearson, et al., 1990) performs a Pearson and Lipman search forsimilarity between a query sequence and a group of sequences of the sametype (nucleic acid or protein); “TfastA” performs a Pearson and Lipmansearch for similarity between a protein query sequence and any group ofnucleotide sequences (it translates the nucleotide sequences in all sixreading frames before performing the comparison); “FastX” performs aPearson and Lipman search for similarity between a nucleotide querysequence and a group of protein sequences, taking frameshifts intoaccount. “TfastX” performs a Pearson and Lipman search for similaritybetween a protein query sequence and any group of nucleotide sequences,taking frameshifts into account (it translates both strands of thenucleic sequence before performing the comparison).

Modified Proteins

The invention encompasses fragments of proteins comprising at least onefollistatin domain. Such fragments will likely include all or a part ofthe follistatin domain. Fragments may include all, a part, or none ofthe sequences between the follistatin domain and the N-terminus and/orbetween the follistatin domain and the C-terminus.

It is understood by one of ordinary skill in the art that certain aminoacids may be substituted for other amino acids in a protein structurewithout adversely affecting the activity of the protein, e.g., bindingcharacteristics of a protein comprising at least one follistatin domain.It is thus contemplated by the inventors that various changes may bemade in the amino acid sequences of proteins comprising at least onefollistatin domain, or DNA sequences encoding the proteins, withoutappreciable loss of their biological utility or activity. Such changesmay include deletions, insertions, truncations, substitutions, fusions,shuffling of motif sequences, and the like.

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biological function on a protein is generallyunderstood in the art (Kyte and Doolittle (1982) J. Mol. Biol., 157:105-132). It is accepted that the relative hydropathic character of theamino acid contributes to the secondary structure of the resultantprotein, which in turn defines the interaction of the protein with othermolecules, for example, enzymes, substrates, receptors, DNA, antibodies,antigens, and the like.

Each amino acid has been assigned a hydropathic index on the basis ofits hydrophobicity and charge characteristics (Kyte and Doolittle,1982); these are isoleucine (+4.5), valine (+4.2), leucine (+3.8),phenylalanine (+2.8), cysteine/cystine (+2.5), methionine (+1.9),alanine (+1.8), glycine (−0.4), threonine (−0.7), serine (−0.8),tryptophan (−0.9), tyrosine (−1.3), proline (−1.6), histidine (−3.2),glutamate (−3.5), glutamine (−3.5), aspartate (−3.5), asparagine (−3.5),lysine (−3.9), and arginine (−4.5). In making such changes, thesubstitution of amino acids whose hydropathic indices may be within ±2,within ±1, and within ±0.5.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101 states that the greatest local average hydrophilicity of aprotein, as governed by the hydrophilicity of its adjacent amino acids,correlates with a biological property of the protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0),lysine (+3.0), aspartate (+3.0±1), glutamate (+3.0±1), serine (+0.3),asparagine (+0.2), glutamine (+0.2), glycine (0), threonine (−0.4),proline (−0.5+1), alanine (−0.5), histidine (−0.5), cysteine (−1.0),methionine (−1.3), valine (−1.5), leucine (−1.8), isoleucine (−1.8),tyrosine (−2.3), phenylalanine (−2.5), and tryptophan (−3.4). In makingsuch changes, the substitution of amino acids whose hydrophilicityvalues may be within ±2, within ±1, and within ±10.5.

The modifications may be conservative such that the structure orbiological function of the protein is not affected by the change. Suchconservative amino acid modifications are based on the relativesimilarity of the amino acid side-chain substituents, for example, theirhydrophobicity, hydrophilicity, charge, size, and the like. Exemplaryconservative substitutions which take various of the foregoingcharacteristics into consideration are well known to those of skill inthe art and include: arginine and lysine; glutamate and aspartate;serine and threonine; glutamine and asparagine; and valine, leucine, andisoleucine. The amino acid sequence of proteins comprising at least onefollistatin domain may be modified to have any number of conservativechanges, so long as the binding of the protein to its target antigen isnot adversely affected. Such changes may be introduced inside or outsideof the binding portion of the protein comprising at least onefollistatin domain. For example, changes introduced inside of theantigen binding portion of the protein may be designed to increase theaffinity of the protein for its target.

Stabilizing Modification

Stabilizing modifications are capable of stabilizing a protein,enhancing the in vitro and/or in vivo half life of a protein, enhancingcirculatory half life of a protein and/or reducing proteolyticdegradation of a protein. Such stabilizing modifications include but arenot limited to fusion proteins, modification of a glycosylation site,and modification of carbohydrate moiety. A stabilizer protein may be anyprotein which enhances the overall stability of the modified GDFpropeptide. As will be recognized by one of ordinary skill in the art,such fusion protein may optionally comprise a linker peptide between thepropeptide portion and the stabilizing portion. As is well known in theart, fusion proteins are prepared such that the second protein is fusedin frame with the first protein such that the resulting translatedprotein comprises both the first and second proteins. For example, inthe present invention, a fusion protein may be prepared such that aprotein comprising at least one follistatin domain is fused to a secondprotein (e.g. a stabilizer protein portion.) Such fusion protein isprepared such that the resulting translated protein contains both thepropeptide portion and the stabilizer portion.

Proteins comprising at least one follistatin domain can be glycosylatedor linked to albumin or a nonproteineous polymer. For instance, proteinscomprising at least one follistatin domain may be linked to one of avariety of nonproteinaceous polymers, e.g., polyethylene glycol,polypropylene glycol, or polyoxyalkylenes, in the manner set forth inU.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or4,179,337. Proteins are chemically modified by covalent conjugation to apolymer to increase their circulating half-life, for example. Polymers,and methods to attach them to peptides, are also shown in U.S. Pat. Nos.4,766,106; 4,179,337; 4,495,285; and 4,609,546.

Proteins comprising at least one follistatin domain may be pegylated.Pegylation is a process whereby polyethylene glycol (PEG) is attached toa protein in order to extend the half-life of the protein in the body.Pegylation of proteins comprising at least one follistatin domain maydecrease the dose or frequency of administration of the proteins neededfor an optimal inhibition of GDF-8. Reviews of the technique areprovided in Bhadra et al. (2002) Pharmazie, 57: 5-29, and in Harris etal. (2001) Clin. Pharmacokinet., 40: 539-551.

Proteins comprising at least one follistatin domain can be linked to anFc region of an IgG molecule. Proteins comprising at least onefollistatin domain may be fused adjacent to the Fc region of the IgGmolecule, or attached to the Fc region of the IgG molecule via a linkerpeptide. Use of such linker peptides is well known in the proteinbiochemistry art. The Fc region may me derived from IgG1 or IgG4, forexample.

Proteins comprising at least one follistatin domain may be modified tohave an altered glycosylation pattern (i.e., altered from the originalor native glycosylation pattern). As used herein, “altered” means havingone or more carbohydrate moieties deleted, and/or having one or moreglycosylation sites added to the original protein.

Glycosylation of proteins is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to proteins comprising at least onefollistatin domain is conveniently accomplished by altering the aminoacid sequence such that it contains one or more of the above-describedtripeptide sequences (for N-linked glycosylation sites). The alterationmay also be made by the addition of, or substitution by, one or moreserine or threonine residues to the sequence of the original protein(for O-linked glycosylation sites). For ease, the protein amino acidsequence may be altered through changes at the DNA level.

Another means of increasing the number of carbohydrate moieties onproteins is by chemical or enzymatic coupling of glycosides to the aminoacid residues of the protein. These procedures are advantageous in thatthey do not require production of the GDF peptide inhibitor in a hostcell that has glycosylation capabilities for N- or O-linkedglycosylation. Depending on the coupling mode used, the sugars may beattached to (a) arginine and histidine, (b) free carboxyl groups, (c)free sulfhydryl groups such as those of cysteine, (d) free hydroxylgroups such as those of serine, threonine, or hydroxyproline, (e)aromatic residues such as those of phenylalanine, tyrosine, ortryptophan, or (f) the amide group of glutamine. These methods aredescribed in WO 87/05330, and in Aplin and Wriston (1981) CRC Crit. Rev.Biochem., 22: 259-306.

Removal of any carbohydrate moieties present on proteins comprising atleast one follistatin domain may be accomplished chemically orenzymatically. Chemical deglycosylation requires exposure of the proteinto trifluoromethanesulfonic acid, or an equivalent compound. Thistreatment results in the cleavage of most or all sugars except thelinking sugar (N-acetylglucosamine or N-acetylgalactosamine), whileleaving the amino acid sequence intact.

Chemical deglycosylation is described by Hakimuddin et a/. (1987) Arch.Biochem. Biophys., 259: 52; and Edge et al. (1981) Anal. Biochem.,118:131. Enzymatic cleavage of carbohydrate moieties on GDF peptideinhibitors can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al. (1987) Meth. Enzymol.,138: 350.

Proteins comprising at least one follistatin domain may be linked to theprotein albumin or a derivative of albumin. Methods for linking proteinsand polypeptides to albumin or albumin derivatives are well known in theart. See, for example, U.S. Pat. No. 5,116,944.

Pharmaceutical Compositions

The present invention provides compositions containing proteinscomprising at least one follistatin domain. Such compositions may besuitable for pharmaceutical use and administration to patients. Thecompositions typically contain one or more proteins comprising at leastone follistatin domain and a pharmaceutically acceptable excipient. Asused herein, the phrase “pharmaceutically acceptable excipient” includesany and all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and thelike, that are compatible with pharmaceutical administration. The use ofsuch media and agents for pharmaceutically active substances is wellknown in the art. The compositions may also contain other activecompounds providing supplemental, additional, or enhanced therapeuticfunctions. The pharmaceutical compositions may also be included in acontainer, pack, or dispenser together with instructions foradministration.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Methods toaccomplish the administration are known to those of ordinary skill inthe art. The administration may, for example, be intravenous,intramuscular, or subcutaneous.

Solutions or suspensions used for subcutaneous application typicallyinclude one or more of the following components: a sterile diluent suchas water for injection, saline solution, fixed oils, polyethyleneglycols, glycerine, propylene glycol or other synthetic solvents;antibacterial agents such as benzyl alcohol or methyl parabens;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetra acetic acid; buffers such as acetates,citrates or phosphates; and agents for the adjustment of tonicity suchas sodium chloride or dextrose. The pH can be adjusted with acids orbases, such as hydrochloric acid or sodium hydroxide. Such preparationsmay be enclosed in ampoules, disposable syringes or multiple dose vialsmade of glass or plastic.

Pharmaceutical compositions suitable for injection include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersion. For intravenous administration, suitable carriers includephysiological saline, bacteriostatic water, Cremophor EL™ (BASF,Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, thecomposition must be sterile and should be fluid to the extent that easysyringability exists. It must be stable under the conditions ofmanufacture and storage and must be preserved against the contaminatingaction of microorganisms such as bacteria and fungi. The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (for example, glycerol, propylene glycol, and liquidpolyetheylene glycol, and the like), and suitable mixtures thereof. Theproper fluidity can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. Prevention of theaction of microorganisms can be achieved by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol,ascorbic acid, thimerosal, and the like. In many cases, one may includeisotonic agents, for example, sugars, polyalcohols such as manitol,sorbitol, sodium chloride in the composition. Prolonged absorption ofthe injectable compositions can be brought about by including in thecomposition an agent which delays absorption, for example, aluminummonostearate and gelatin.

In one embodiment, proteins comprising at least one follistatin domainare prepared with carriers that will protect the compound against rapidelimination from the body, such as a controlled release formulation,including implants and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Methods for preparation of suchformulations will be apparent to those skilled in the art. The materialscan also be obtained commercially from Alza Corporation and NovaPharmaceuticals, Ind. Liposomal suspensions containing proteinscomprising at least one follistatin domain can also be used aspharmaceutically acceptable carriers. These can be prepared according tomethods known to those skilled in the art, for example, as described inU.S. Pat. No. 4,522,811.

Therapeutically useful agents, such as growth factors (e.g., BMPs,TGF-β, FGF, IGF), cytokines (e.g., interleukins and CDFs), antibiotics,and any other therapeutic agent beneficial for the condition beingtreated may optionally be included in or administered simultaneously orsequentially with, proteins comprising at least one follistatin domain.

It is especially advantageous to formulate compositions in dosage unitform for ease of administration and uniformity of dosage. Dosage unitform as used herein refers to physically discrete units suited asunitary dosages for the subject to be treated; each unit containing apredetermined quantity of active compound calculated to produce thedesired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on the uniquecharacteristics of the active compound and the particular therapeuticeffect to be achieved, and the limitations inherent in the art ofcompounding such an active compound for the treatment of individuals.

Treatment Indications

Proteins comprising at least one follistatin domain are useful toprevent, diagnose, or treat various medical disorders in humans oranimals. Accordingly, the present invention provides a method fortreating diseases and disorders related to muscle cells and tissue, byadministering to a subject a composition comprising at least one proteincomprising at least one follistatin domain in an amount sufficient toameliorate the symptoms of the disease. Such disorders include musculardystrophies, including, but not limited to severe or benign X-linkedmuscular dystrophy, limb-girdle dystrophy, facioscapulohumeraldystrophy, myotinic dystrophy, distal muscular dystrophy, progressivedystrophic ophthalmoplegia, oculopharyngeal dystrophy, Duchenne'smuscular dystrophy, and Fakuyama-type congenital muscular dystophy);amyotrophic lateral sclerosis (ALS); muscle atrophy; organ atrophy;frailty; carpal tunnel syndrome; congestive obstructive pulmonarydisease; congenital myopathy; myotonia congenital; familial periodicparalysis; paroxysmal myoglobinuria; myasthenia gravis; Eaton-Lambertsyndrome; secondary myasthenia; denervation atrophy; paroxymal muscleatrophy; and sarcopenia, cachexia and other muscle wasting syndromes.The invention also relates to traumatic or chronic injury to muscletissue.

In addition to providing therapy for muscle diseases and disorders, thepresent invention also provides for methods for preventing or treatingmetabolic diseases or disorders resulting from abnormal glucosehomeostasis. Such diseases or disorders include metabolic diseases anddisorders (such as insulin-dependent (type 1) diabetes mellitus,noninsulin-dependent (type 2) diabetes mellitus), hyperglycemia,impaired glucose tolerance, metabolic syndrome (e.g., syndrome X),obesity and insulin resistance induced by trauma (e.g., burns ornitrogen imbalance), adipose tissue disorders (such as obesity), or bonedegenerative diseases (such as osteoporosis, especially in the elderlyand/or postmenopausal women; glucocorticoid-induced osteoporosis;osteopenia; osteoarthritis; and osteoporosis-related fractures). Yetfurther examples include low bone mass due to chronic glucocorticoidtherapy, premature gonadal failure, androgen suppression, vitamin Ddeficiency, secondary hyperparathyroidism, nutritional deficiencies, andanorexia nervosa.

Normal glucose homeostasis requires the finely tuned orchestration ofinsulin secretion by pancreatic beta cells in response to subtle changesin blood glucose levels. One of the fundamental actions of insulin is tostimulate uptake of glucose from the blood into tissues, especiallymuscle and fat.

Accordingly, the present invention provides a method for treatingdiabetes mellitus and related disorders, such as obesity orhyperglycemia, by administering to a subject a composition comprising atleast one protein comprising at least one follistatin domain in anamount sufficient to ameliorate the symptoms of the disease. Type 2 ornoninsulin-dependent diabetes mellitus (NIDDM), in particular, ischaracterized by a triad of (1) resistance to insulin action on glucoseuptake in peripheral tissues, especially skeletal muscle and adipocytes,(2) impaired insulin action to inhibit hepatic glucose production, and(3) dysregulated insulin secretion (DeFronzo (1997) Diabetes Rev. 5:177-269). Therefore, subjects suffering from type 2 diabetes can betreated according to the present invention by administration of proteincomprising at least one follistatin domain, which increases sensitivityto insulin and glucose uptake by cells.

Similarly, other diseases and metabolic disorders characterized byinsulin dysfunction (e.g., resistance, inactivity, or deficiency) and/orinsufficient glucose transport into cells also can be treated accordingto the present invention by administration of a protein comprising atleast one follistatin domain, which increases sensitivity to insulin andglucose uptake by cells.

Methods of Treatment Using Proteins

Proteins comprising at least one follistatin domain may be used toinhibit or reduce one or more activities associated with the GDF-8protein (whether in monomeric form, dimeric active form, or complexed ina GDF-8 latent complex), relative to a GDF-8 protein not bound by thesame protein. In an embodiment, the activity of the mature GDF-8protein, when bound by a protein comprising at least one follistatindomain, is inhibited at least 50%, or at least 60, 62, 64, 66, 68, 70,72, 72, 76, 78, 80, 82, 84, 86, or 88%, or at least 90, 91, 92, 93, or94%, or at least 95% to 100% relative to a mature GDF-8 protein that isnot bound by a protein having a follistatin domain.

Pharmaceutical preparations comprising proteins comprising at least onefollistatin domain are administered in therapeutically effectiveamounts. As used herein, an “effective amount” of the protein is adosage which is sufficient to reduce the activity of GDF-8 to achieve adesired biological outcome. The desired biological outcome may be anytherapeutic benefit including an increase in muscle mass, an increase inmuscle strength, improved metabolism, decreased adiposity, or improvedglucose homeostasis. Such improvements may be measured by a variety ofmethods including those that measure lean and fat body mass (such asduel x-ray scanning analysis), muscle strength, serum lipids, serumleptin, serum glucose, glycated hemoglobin, glucose tolerance, andimprovement in the secondary complication of diabetes.

Generally, a therapeutically effective amount may vary with thesubject's age, condition, and sex, as well as the severity of themedical condition in the subject. The dosage may be determined by anphysician and adjusted, as necessary, to suit observed effects of thetreatment. Appropriate dosages for administering at least one proteincomprising at least one follistatin domain may range from 5 mg to 100mg, from 15 mg to 85 mg, from 30 mg to 70 mg, or from 40 mg to 60 mg.Proteins can be administered in one dose, or at intervals such as oncedaily, once weekly, and once monthly. Dosage schedules can be adjusteddepending on the affinity of the protein for GDF-8, the half life of theprotein, or the severity of the patient's condition. Generally, thecompositions are administered as a bolus dose, to maximize thecirculating levels of proteins comprising at least one follistatindomain for the greatest length of time after the dose. Continuousinfusion may also be used after the bolus dose.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Proteins comprising at least one follistatin domain which exhibit largetherapeutic indices may be used.

Data obtained from the cell culture assays and animal studies can beused in evaluating a range of dosage for use in humans. The dosage ofsuch compounds may lie within a range of circulating concentrations thatinclude the ED₅₀ with little or no toxicity. The dosage may vary withinthis range depending upon the dosage form employed and the route ofadministration utilized. For any protein comprising at least onefollistatin domain used in the present invention, the therapeuticallyeffective dose can be estimated initially from cell culture assays. Adose may be formulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test protein which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Levels in plasma may be measured, forexample, by high performance liquid chromatography. The effects of anyparticular dosage can be monitored by a suitable bioassay. Examples ofsuitable bioassays include GDF-8 protein/receptor binding assays,creatine kinase assays, assays based on glucose uptake in adipocytes,and immunological assays.

Methods of Administering DNA

The present invention also provides gene therapy for the in vivoproduction of proteins comprising at least one follistatin domain. Suchtherapy would achieve its therapeutic effect by introduction of thepolynucleotide sequences into cells or tissues having the disorders aslisted herein.

Delivery of polynucleotide sequences of proteins comprising at least onefollistatin domain can be achieved using a recombinant expression vectorsuch as a chimeric virus or a colloidal dispersion system. Targetliposomes may be used for therapeutic delivery of the polynucleotidesequences. Various viral vectors which can be utilized for gene therapyinclude adenovirus, herpes virus, vaccinia, or an RNA virus such as aretrovirus. The retroviral vector may be a derivative of a murine oravian retrovirus. Examples of retroviral vectors in which a singleforeign gene can be inserted include, but are not limited to: Moloneymurine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV),murine mammary tumor virus (MuMTV), and Rous sarcoma virus (RSV). Anumber of additional retroviral vectors can incorporate multiple genes.All of these vectors can transfer or incorporate a gene for a selectablemarker so that transduced cells can be identified and generated. Byinserting a GDF propeptide polynucleotide sequence of interest into theviral vector, along with another gene which encodes the ligand for areceptor on a specific target cell, for example, the vector is nowtarget specific.

Retroviral vectors can be made target specific by attaching, forexample, a sugar, a glycolipid, or a protein. Targeting may beaccomplished by using an antibody. Those of skill in the art willrecognize that specific polynucleotide sequences can be inserted intothe retroviral genome or attached to a viral envelope to allow targetspecific delivery of the retroviral vector containing the polynucleotideof proteins comprising at least one follistatin domain. In oneembodiment, the vector is targeted to muscle cells or muscle tissue.

Since recombinant retroviruses are defective, they require helper celllines that contain plasmids encoding all of the structural genes of theretrovirus under the control of regulatory sequences within the LTR.These plasmids are missing a nucleotide sequence which enables thepackaging mechanism to recognize an RNA transcript for encapsidation.Helper cell lines which have deletions of the packaging signal include,but are not limited to PSI.2, PA317 and PA12, for example. These celllines produce empty virions, since no genome is packaged. If aretroviral vector is introduced into such cells in which the packagingsignal is intact, but the structural genes are replaced by other genesof interest, the vector can be packaged and vector virion produced.

Alternatively, other tissue culture cells can be directly transfectedwith plasmids encoding the retroviral structural genes gag, pol and env,by conventional calcium phosphate transfection. These cells are thentransfected with the vector plasmid containing the genes of interest.The resulting cells release the retroviral vector into the culturemedium.

Another targeted delivery system for a polynucleotide of a proteincomprising at least one follistatin domain is a colloidal dispersionsystem. Colloidal dispersion systems include macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes.Liposomes are artificial membrane vesicles which are useful as deliveryvehicles in vitro and in vivo. RNA, DNA and intact virions can beencapsulated within the aqueous interior and be delivered to cells in abiologically active form (see, for example, Fraley, et al. (1981) TrendsBiochem. Sci., 6: 77). Methods for efficient gene transfer using aliposome vehicle, are known in the art (see, for example, Mannino, etal. (1988) Biotechniques, 6: 682. The composition of the liposome isusually a combination of phospholipids, usually in combination withsteroids, especially cholesterol. Other phospholipids or other lipidsmay also be used. The physical characteristics of liposomes depend onpH, ionic strength, and the presence of divalent cations.

Examples of lipids useful in liposome production include phosphatidylcompounds, such as phosphatidylglycerol, phosphatidylcholine,phosphatidylserine, phosphatidylethanolamine, sphingolipids,cerebrosides, and gangliosides. Illustrative phospholipids include eggphosphatidylcholine, dipalmitoylphosphatidylcholine anddistearoylphosphatidylcholine. The targeting of liposomes is alsopossible based on, for example, organ-specificity, cell-specificity, andorganelle-specificity and is known in the art.

There is a wide range of methods which can be used to deliver the cellsexpressing proteins comprising at least one follistatin domain to a sitefor use in modulating a GDF-8 response. In one embodiment of theinvention, the cells expressing follistatin protein can be delivered bydirect application, for example, direct injection of a sample of suchcells into the site of tissue damage. These cells can be purified. Thesuch cells can be delivered in a medium or matrix which partiallyimpedes their mobility so as to localize the cells to a site of injury.Such a medium or matrix could be semi-solid, such as a paste or gel,including a gel-like polymer. Alternatively, the medium or matrix couldbe in the form of a solid, a porous solid which will allow the migrationof cells into the solid matrix, and hold them there while allowingproliferation of the cells.

Methods of Detection and Isolation of GDF-8

Proteins comprising at least one follistatin domain may be used todetect the presence or level of GDF-8, in vivo or in vitro. Bycorrelating the presence or level of these proteins with a medicalcondition, one of skill in the art can diagnose the associated medicalcondition. The medical conditions that may be diagnosed by the proteinscomprising at least one follistatin domain are set forth herein.

Such detection methods are well known in the art and include ELISA,radioimmunoassay, immunoblot, western blot, immunofluorescence,immunoprecipitation, and other comparable techniques. Proteinscomprising at least one follistatin domain may further be provided in adiagnostic kit that incorporates one or more of these techniques todetect GDF-8. Such a kit may contain other components, packaging,instructions, or other material to aid the detection of the protein anduse of the kit.

Where proteins comprising at least one follistatin domain are intendedfor diagnostic purposes, it may be desirable to modify them, for examplewith a ligand group (such as biotin) or a detectable marker group (suchas a fluorescent group, a radioisotope or an enzyme). If desired, theproteins may be labeled using conventional techniques. Suitable labelsinclude fluorophores, chromophores, radioactive atoms, electron-densereagents, enzymes, and ligands having specific binding partners. Enzymesare typically detected by their activity. For example, horseradishperoxidase is usually detected by its ability to convert3,3′,5,5′-tetramethylbenzidine (TMB) to a blue pigment, quantifiablewith a spectrophotometer. Other suitable binding partners include biotinand avidin or streptavidin, IgG and protein A, and the numerousreceptor-ligand couples known in the art. Other permutations andpossibilities will be readily apparent to those of ordinary skill in theart, and are considered as equivalents within the scope of the instantinvention.

Proteins comprising at least one follistatin domain or fragments thereofmay also be useful for isolating GDF-8 in a purification process. In onetype of process, proteins may be immobilized, for example, throughincorporation into a column or resin. The proteins are used to bindGDF-8, and then subject to conditions which result in the release of thebound GDF-8. Such processes may be used for the commercial production ofGDF-8.

The following examples provide embodiments of the invention. One ofordinary skill in the art will recognize the numerous modifications andvariations that may be performed without altering the spirit or scope ofthe present invention. Such modifications and variations are believed tobe encompassed within the scope of the invention. The examples do not inany way limit the invention. It is understood that all of the numbers inthe specification and claims are modified by the term about, as smallchanges in dosages, for example, would be considered to be within thescope of the invention.

EXAMPLES Example 1 Purification of JA16-Conjugated Beads

N-hydroxysuccinimidyl-activated beads (4% beaded agarose, Sigma H-8635,St Louis Mo.) were washed in MilliQ-H₂O and incubated for 4 hours at 4°C. with the anti-GDF-8 JA16 monoclonal antibody (3-4 μg/μl in 100 mMMOPS, pH 7.5) at a ratio to allow a final concentration of 10 mg JA16/mlresin. Beads were washed extensively with 100 mM MOPS pH 7.5 andphosphate-buffered saline (PBS) (Ausubel et al, (1999) Current Protocolsin Molecular Biology, John Wiley & Sons) and stored at 4° C. in PBSuntil use. Control beads were prepared identically without JA16antibody.

Example 2 Affinity Purification

A total of 40 μl of packed JA16-conjugated or control beads wereincubated with 15 ml normal Balb/C mouse serum (Golden West Biologicals,Temecula Calif.) or 30 ml pooled normal human serum (ICN Biomedical,Aurora Ohio) for 3 hours at 4° C. Beads were washed twice in ˜10 ml ofcold 1% Triton X-100/PBS, twice in ˜10 ml of cold 0.1% Triton X-100/PBS,and twice in ˜1 ml of cold PBS. Proteins were eluted from the beads inthree subsequent steps. First, the beads were treated to a ‘mockelution’, where 100 μl of PBS was added to the beads and incubated at 4°C. for 30 minutes. The supernatant was collected and combined with 30 μl4×LDS sample buffer (Invitrogen, Carlsbad Calif.). Second, the beadswere subject to a ‘peptide elution’, 100 μl of 1 μg/μl competing peptide(sequence: DFGLDSDEHSTESRSSRYPLTVDFEAFGWDCOOH (SEQ ID NO:12)) in PBS wasadded to the beads and again incubated at 4° C. for 30 minutes. Thesupernatant was collected as before. Third, the beads were treated withan ‘SDS elution’ technique, where 30 μl of 4×LDS buffer (Invitrogen) and100 μl of PBS was added to the beads and heated to 80° C. for 10 minutesbefore transferring the supernatant to a fresh tube.

A silver stained gel of the proteins released in each of the elutionsteps is shown in FIG. 1. Two protein bands in the silver-stained gelshown in FIG. 1 of approximately 12 and 36 kDa were specifically elutedfrom JA16-conjugated beads, but not from unconjugated control beads.

Example 3 Mass Spectrometry

Samples were reduced with NuPage 10×reducing agent (Invitrogen) for 10minutes at 80° C. and alkylated with 110 μM iodoacetamide for 30 minutesat 22° C. in the dark. Samples were run immediately on 10% NuPageBis-Tris gels in an MES buffer system according to manufacturer'srecommendations (Invitrogen) and silver stained using agluteraldehyde-free system (Shevchenko, et a., (1996) Anal. Chem., 68:850-858). Bands were excised and digested with Sequencing Grade ModifiedTrypsin (Promega, Madison Wis.) in an Abimed Digest Pro (Langenfeld,Germany) or ProGest Investigator (Genomics Solutions, Ann Arbor Mich.).The volume of digested samples was reduced by evaporation andsupplemented with 1% acetic acid to a final volume of ˜20 μl. Samples(5-10 μl) were loaded onto a 10 cm×75 μm inner diameter C₁₈ reversephase column packed in a Picofrit needle (New Objectives, Woburn Mass.).MS/MS data was collected using an LCQ Deca or LCQ Deca XP (Finnigan, SanJose Calif.) mass spectrometer and searched against the NCBInon-redundant database using the Sequest program (Finnigan). Unlessotherwise noted, all peptide sequences listed in this paper correspondedto MS/MS spectra that were deemed high quality by manual inspection andproduced X_(corr) scores>2.5 in the Sequest scoring system.

Example 4 Western Blots

Proteins were transferred to a 0.45 μm nitrocellulose membrane(Invitrogen) and blocked with blocking buffer (5% non-fat dry milk inTris-buffered saline (TBS: 10 mM Tris-Cl, pH 7.5, 150 mM NaCl)) at 4° C.overnight. Blots were then probed with primary antibody diluted 1:1000in blocking buffer for 1-3 hours at room temperature, washed 5×with TBS,probed with horseradish peroxidase-conjugated secondary antibody inblocking buffer for 1-3 hours at room temperature, and washed as before.Signals were detected by autoradiography using the West Pico Substrate(Pierce).

Example 5 Isolation of GDF-8

An experiment using the methods described in the previous Examplesresulted in the isolation of GDF-8. Since GDF-8 in its reduced form is12 kDa, we speculated that the protein in the lower band from thesilver-stained gel shown in FIG. 1 was mature GDF-8. To confirm thishypothesis, we excised this band, digested it with trypsin, and obtainedMS/MS spectra of the resulting peptides by LC-MS/MS. MS/MS spectracorresponding to six tryptic peptides confirmed that mature GDF-8 wasisolated from this region of the gel, as shown in FIG. 2A and Table 1.

Table 1 lists peptides derived from GDF-8 (SEQ ID NO:13-20), GDF-8propeptide (SEQ ID NO:21-27), FLRG (SEQ ID NO:28-30), and GASP1 (SEQ IDNO:31-35) that were found in JA16 immunoprecipitates from mouse andhuman serum. The immediately preceding amino acid in the proteinsequence is shown in parentheses for each peptide and the charge stateof the peptide (z) and the Sequest program correlation coefficient(X_(corr), a measure of confidence) are listed. The sequence listingnumbers in the table refer only to the isolated peptides and theirsequences. The preceding amino acids in parentheses are not included inthe peptides, but are provided only for reference. All spectra wereconfirmed by manual inspection.

Interestingly, the western blot also contained a band corresponding tounprocessed full-length GDF-8 (43 kDa), implying that some portion ofthis molecule is secreted into serum without undergoing proteolyticprocessing (FIG. 2B). The presence of unprocessed GDF-8 was confirmed mymass spectrometry (data not shown). Thus, the affinity purificationmethod effectively isolated GDF-8 from normal mouse serum.

Although the JA16 antibody recognizes both GDF-8 and the highly relatedprotein BMP/GDF-11, we saw no evidence of BMP-11 peptides in ouraffinity purified samples by mass spectrometry.

Table 1: Peptides Identified in JA16 Immunoprecipitates

TABLE 1 Peptides Identified in JA16 Immunoprecipitates z X_(corr) mouseserum GDF-8 (K) ANYCSGECEFVFLQK (SEQ ID NO:13) 3+ 4.63 (mature) (K)MSPINMLYFNGK (SEQ ID NO:14) 2+ 3.81 (R) DFGLDCDEHSTESR (SEQ ID NO:15) 2+3.47 (K) ANYCSGECEFVFLQK (SEQ ID NO:16) 2+ 3.31 (K) M*SPINMLYFNGK (SEQID NO:17) 3+ 2.95 (R) YPLTVDFEAFGWDWIIAPK (SEQ ID NO:18) 2+ 2.86 (K)M*SPINM*LYFNGK (SEQ ID NO:19) 2+ 2.51 (R) GSAGPCCTPTK (SEQ ID NO:20) 2+2.43 GDF-8 (K) LDM*SPGTGIWQSIDVK (SEQ ID NO:21) 2+ 3.82 (propeptide) (K)ALDENGHDLAVTFPGPGEDGLNPFLEVK (SEQ ID NO:22) 3+ 3.17 (K) LDMSPGTGIWQSIDVK(SEQ ID NO:23) 2+ 2.98 (R) ELIDQYDVQR (SEQ ID NO:24) 2+ 2.97 (K)TPTTVFVQILR (SEQ ID NO:25) 2+ 2.91 (K) AQLWIYLRPVK (SEQ ID NO:26) 2+2.77 (K) EGLCNACAWR (SEQ ID NO:27) 2+ 2.75 follistatin-like (R)PQSCLVDQTGSAHCVVCR (SEQ ID NO:28) 3+ 3.34 related gene FLRG (K)DSCDGVECGPGK (SEQ ID NO:29) 2+ 2.99 (K) SCAQVVCPR (SEQ ID NO:30) 2+ 2.59novel (R) ECETDQECETYEK (SEQ ID NO:31) 2+ 2.98 multidomain protease (R)ADFPLSVVR (SEQ ID NO:32) 2+ 2.56 inhibitor (GASP1) (R) EACEESCPFPR (SEQID NO:33) 2+ 2.95 (R) SDFVILGR (SEQ ID NO:34) 2+ 2.73 (R) VSELTEEQDSGR(SEQ ID NO:35) 2+ 3.88 human serum GDF-8 (K) ANYCSGECEFVFLQK (SEQ IDNO:36) 2+ 4.21 mature (R) DFGLDCDEHSTESR (SEQ ID NO:37) 3+ 2.08 GDF-8(K) ALDENGHDLAVTFPGPGEDGLNPFLEVK (SEQ ID NO:38) 3+ 3.71 (propeptide) (R)ELIDQYDVQR (SEQ ID NO:39) 2+ 3.01 follistatin-like (R)PQSCVVDQTGSAHCVVCR (SEQ ID NO:40) 3+ 3.37 related gene FLRG (R)CECAPDCSGLPAR (SEQ ID NO:41) 2+ 3.21 (R) LQVCGSDGATYR (SEQ ID NO:42) 2+3.06 multidomain (R) VSELTEEPDSGR (SEQ ID NO:43) 2+ 2.44 proteaseinhibitor (R) CYMDAEACSK (SEQ ID NO:44) 2+ 2.69 (GASP1) (K) GITLAVVTCR(SEQ ID NO:45) 2+ 2.42 M* = oxidized methionine

Example 6 Isolation of Proteins Bound to GDF-8

Once it was confirmed that the affinity purification technique couldsuccessfully isolate GDF-8 from normal mouse serum, we proceeded toidentify proteins that bind to GDF-8 under native conditions. The 36 kDaband on the silver-stained gel shown in FIG. 1 was analyzed as describedabove. Mass spectrometry identified two proteins in this region of thegel that were specific to the JA16-immunopurified sample. These weredetermined to be the GDF-8 propeptide and follistatin-like related gene(FLRG). The peptides identified from each of these proteins are shown inTable 1 (SEQ ID NO:13-27). High quality MS/MS spectra were found for sixunique peptides from GDF-8 propeptide and three unique peptides fromFLRG; representative peptides are shown in FIGS. 3A and 3C. Furthermore,the presence of both of these proteins was confirmed by western blottingwith polyclonal antibodies specific to GDF-8 propeptide and FLRGrespectively (FIGS. 3B and 3D). Thus, circulating GDF-8 appears to bindto the GDF-8 propeptide and to FLRG in vivo.

Example 7 Isolation of Novel Proteins that Bind GDF-8

To characterize the major components of the circulating GDF-8 complex invivo, native GDF-8 and its associated proteins from wild-type mouseserum were isolated by affinity purification with an agarose-conjugatedanti-GDF-8 monoclonal antibody, JA16. JA16-bound proteins were subjectedto subsequent elution steps with PBS buffer alone (mock elution), apeptide that could compete with GDF-8 for JA16 binding, and SDSdetergent. These samples were concentrated, run on a one-dimensionalSDS-PAGE gel, and visualized by silver stain (FIG. 4). Two bands uniqueto the JA16 purified samples are visible-a 12 kDa band identified asGDF-8, and a 36 kDa band containing both GDF-8 propeptide and FLRG.

In order to determine if one could identify other proteins that werebound to GDF-8 in vivo, we scaled up the purification approximatelyfive-fold and used mass spectrometry to search for proteins that werepresent in the JA16 immunocomplex, but not in the negative control. Toachieve this goal, we excised regions of the silver stained gelcorresponding to molecular weights between 10 and 200 kDa into 13 gelslices, as shown in FIG. 4. Each of these slices was subjected to in-geltrypsin digestion and LC-MS/MS. Comparison of the resulting MS/MSspectra to the non-redundant NCBI database of known proteins did notreveal any additional proteins specific to the JA16 immunoprecipitate,although the proteins previously described (mature GDF-8, GDF-8propeptide, unprocessed GDF-8, and FLRG) were all identified in thesesamples (FIG. 4). Background proteins that were found both in the JA16immunocomplex and in the negative control sample included abundant serumproteins, such as albumin, immunoglobulins, and complement proteins.There was no evidence of other TGF-β superfamily members, including thehighly related protein BMP-11/GDF-11, in the JA16 samples. Thus, theJA16 antibody specifically purified GDF-8 in these experiments.

Interestingly, we found no evidence of follistatin in our GDF-8immunocomplexes, despite the fact that JA16 is capable ofimmunoprecipitating a GDF-8/follistatin complex in vitro (data notshown). Follistatin has been shown to inhibit GDF-8 activity byantagonizing the association of GDF-8 with the ActRIIB receptor (Lee andMcPherron (2001) Proc. Nat. Acad. Sci. U.S.A., 98: 9306-9311). Ourresults suggest that follistatin does not play a major role in theregulation of the activity of the circulating GDF-8 complex under normalconditions.

Since the identification of proteins by this MS/MS procedure isdependent on the content of the database being searched, we furtheranalyzed the data from FIG. 4 by comparing the MS/MS spectra collectedfrom the 13 samples to a database of proteins predicted from the Celeramouse genomic sequence. This analysis identified an additional proteinspecific to the JA16-purified sample, and is hereby referred to asGDF-associated serum protein 1 (GASP1). Since the initial identificationof this protein, this sequence has been added to the NCBI nr database bythe public genome sequencing effort under the accession numbergi|20914039.

Five peptides corresponding to the sequence of GASP1 were identified onthe basis of high-quality MS/MS spectra (Table 1 (SEQ ID NO:31-35); FIG.5A and B). The spectra corresponding to GASP1 peptides were found inband 3, which contains 70-80 kDa proteins. However, a specific bandcorresponding to this protein was not visible, probably due to theabundance of background immunoglobulins and albumin at this area (seeFIG. 4). Sequest X_(corr) scores above 2.3 are generally consideredsignificant for 2⁺ ions. Fortuitously, one of the peptides identified inour experiments (sequence=ECETDQECETYEK (SEQ ID NO:31)) spans thejunction between the two exons that code for this protein, verifying theaccuracy of Celera's gene prediction algorithm in this instance.

The sequences of the GASP1 transcript and protein were predicted priorto the actual cloning of GASP1 (FIG. 6). GASP1 was predicted to be a 571amino acid protein with a predicted molecular mass of 63 kDa. It has aputative signal sequence/cleavage site at its N-terminus and twopossible sites for N-glycosylation at amino acids 314 and 514. Analysisof the GASP1 protein sequence by Pfam and BLAST (according to thetechniques in Altschul et al.(1990) J. Mol. Biol., 215: 403-410; Batemanet al. (2002) Nucleic Acids Res., 30: 276-280) revealed that GASP1contains many conserved domains, including a WAP domain, afollistatin/Kazal domain, an immunoglobulin domain, two tandem Kunitzdomains, and a netrin domain (FIG. 14A). WAP domains, originallyidentified in whey acidic protein, contain 8 cysteines that form afour-disulfide core and are often found in proteins with anti-proteaseactivity (Hennighausen and Sippel (1982) Nucleic Acids Res., 10:2677-2684; Seemuller et al. (1986) FEBS Lett., 199: 43-48). It isbelieved that the follistatin domain mediates the interaction betweenGDF-8 and GASP1. The C-terminal region of follistatin domains contains asimilarity to Kazal serine protease inhibitor domains. In the case ofGASP1, this region is even more closely related to Kazal domains than infollistatin or FLRG, suggesting the possibility that this region mayhave an additional protease inhibitor function. Kunitz domains,originally identified in bovine pancreatic trypsin inhibitor, alsoinhibit serine proteases, thus establishing a likely role for GASP1 inthe regulation of this class of proteins. Furthermore, netrin domainshave been implicated in the inhibition of metalloproteases (Banyai andPatthy, 1999; Mott et al., 2000). Thus, based on the presence of theseconserved regions, GASP1 is likely to inhibit the activity of proteases,perhaps regulating GDF-8 processing or activation of the latent GDF-8complex.

BLAST searches against the mouse Celera transcript database revealed aprotein that has >50% identity with GASP1, referred to here as GASP2.GASP2 contains the same domain structure as GASP1, suggesting that theseproteins define a two member family of multivalent protease inhibitors(FIG. 14B). Interestingly, only peptides corresponding to GASP1, notGASP2, were found in our JA16 purified samples. This result suggeststhat GASP1 and GASP2 likely have different biological specificity. BothGASP1 and GASP2 are conserved in humans (>90% identity with mouse). Thesequence for human GASP1 is now available in the NCBI nr database underthe accession number gi|18652308. Although, the concentration of GDF-8in human serum is considerably lower than that found in mouse serum(Hill et al. (2002) J. Biol. Chem., 277: 40735-40741), the sensitivityof mass spectrometric analysis of proteins allowed us to identify 3peptides corresponding to the human homolog of GASP1 from JA16immunoprecipitations from human serum (Table 1). None of these peptideswere found in the corresponding negative control. Again, there was noevidence of human GASP2 in these experiments. Thus, the interactionbetween GASP1 and GDF-8 is conserved between mouse and human.

GDF-8 is produced nearly exclusively in skeletal muscle. In order todetermine the tissue distribution of GASP1 mRNA, a 551 bp fragment ofGASP1 was amplified from first-strand cDNA produced from a variety ofmouse tissues and staged embryos (FIG. 10). A mouse GASP1 fragment wasamplified from normalized mouse first-strand cDNA panels (Clontech, PaloAlto Calif.) using the Advantage cDNA PCR kit (Clontech) according tothe manufacturer's recommendations (forward primer: 5′TTGGCCACTGCCACCACAATCTCMCCACTT 3′ (SEQ ID NO:46); reverse primer: 5′TCTCAGCATGGCCATGCCGCCGTCGA 3′ (SEQ ID NO:47)). GASP1 appears to befairly widely-expressed, with particularly high expression in skeletalmuscle and heart. Significant expression is also seen in brain, lung,and testis. In contrast, liver and kidney express relatively low levelsof GASP1 mRNA. Developmentally, the level of GASP1 mRNA remains fairlyconstant, perhaps increasing slightly between day 7 and day 11 of mouseembryogenesis.

Example 8 GDF-8 in Human and Mouse Serum

The concentration of GDF-8 in human serum is considerably lower thanthat found in mouse serum. Since GDF-8 has potential as a therapeutictarget, it was a goal to determine the composition of the circulatingGDF-8 complex in humans. This knowledge would determine the validity ofthe mouse model and potentially identify alternative therapeutictargets. Thus, the JA16-based affinity purification of GDF-8 wasrepeated using human serum. Due to the lower level of GDF-8 in humanserum compared with mouse, no bands corresponding to mature GDF-8 andGDF-8 propeptide/FLRG were visualized (FIG. 11A). However, westernblotting with a polyclonal antibody that recognizes the mature region ofGDF-8 revealed the presence of mature and unprocessed GDF-8 in theJA16-purified samples (FIG. 11B).

We took advantage of the high sensitivity of mass spectrometry toidentify proteins that co-purified with mature GDF8. The lanescorresponding to peptide eluted samples from both negative control andJA16-conjugated beads were sliced into 16 pieces. These gel slices weresubjected to in-gel trypsin digestion, nanoflow LC-MS/MS, and analysiswith Sequest as before.

Interestingly, the only proteins that were identified specifically inthe JA16 samples and not the negative control were mature GDF-8, GDF-8propeptide, human FLRG, and the human homolog of GASP1. The peptidesfound from each of these proteins are listed in Table 1 (SEQ ID NO:3645)and representative MS/MS spectra are shown in FIG. 12. Thus the in vivoGDF-8 complex appears to be conserved between mouse and human.

Example 9 Cloning and Characterization of Mouse GASP1

After identifying the predicted GASP1 sequence, it was a goal todetermine the actual sequence of mouse GASP1. Based on the Celerapredicted sequence, the GASP1 coding sequence was amplified from mouseheart QUICKCLONE cDNA (Clontech) by PCR with PfuTurbo polymerase(Stratagene) using the following primers (fp:5′CACCATGTGTGCCCCAGGGTATCATCGGTTCTGG 3′ (SEQ ID NO:50); rp: 5′TTGCAAGCCCAGGAAGTCCTTGAGGAC 3′ (SEQ ID NO:51)). The PCR product fromthis reaction ran as a single major band of approximately 1700 basepairs on a 1% agarose gel. The amplified DNA was then cloned into theTOPO sites of the pcDNA3.1 D/V5-His-TOPO vector (Invitrogen) so as toinclude an in-frame C-terminal V5-His tag according to manufacturers'recommendations. The full-length cDNA insert was sequenced on bothstrands. The nucleotide sequence of the mouse GASP1 clone is shown inFIG. 13. This clone matched the predicted Celera sequence, with theexception of some base substitutions in wobble codons that did notchange the predicted amino acid sequence (i.e., 288C:G; 294G:A; 615G:A;738A:G; 768C:T; 1407A:G; 1419A:G; and 1584C:G, where the first base atthe indicated position is that reported by Celera and the second base isthat obtained from sequencing of the clone; see FIG. 6A and B).

To determine the N-terminal processing of the GASP1 protein, wetransfected COS1 cells with a mammalian expression vector encoding mouseGASP1 cloned with a C-terminal V5-His tag (GASP1-V5-His). Serum-freeconditioned media was harvested 48 hours later and analyzed by westernblot analysis with an anti-V5 polyclonal antibody (Sigma). Morespecifically, conditioned media was collected 48 hours aftertransfection of COS1 cells with GASP1-V5-His/pcDNA3.1D-V5-His-TOPO orempty vector using the FuGENE 6 reagent (Roche) in serum-free Dulbecco'smodified Eagle's medium.

A single band, running at approximately 80 kDa was seen, confirming thatGASP1 is secreted into the conditioned media (data not shown).Approximately 10 ml of this conditioned media was run over aHis-affinity column and further purified by reverse phasechromatography. This purification scheme yielded a band the expectedsize of full-length GASP1 on a Coomassie stained SDS-PAGE gel. Edmansequencing of this band determined an N-terminal sequence ofL-P-P-I-R-Y-S-H-A-G-I (SEQ ID NO:52). Thus, amino acids 1-29 of GASP1constitute the signal sequence that is removed during processing andsecretion.

Example 10 Binding of Recombinantly-Produced GASP1 to GDF-8 Propeptideand Mature GDF-8

Next, it was determined that recombinantly-produced GASP1 had the samebinding pattern to GDF-8 as GASP1 isolated from mouse serum. Forimmunoprecipitations with recombinant proteins, 400 μl conditioned mediafrom vector- or GASP1-transfected cells was combined with 1.2 μg ofrecombinant purified GDF-8 and/or GDF-8 propeptide protein (Thies etal., 2001). JA16 (10 μl packed volume) or anti-V5 (30 μl) conjugatedagarose beads were incubated with the supplemented conditioned media fortwo hours at 4° C. and washed twice in cold 1% Triton inphosphate-buffered saline (PBS) and twice in PBS. Beads were resuspendedin 50 μl 1×LDS buffer with DTT. Western blots were performed aspreviously described (Hill et al., 2002).

To confirm and further characterize the interaction between GDF-8 andGASP1, we incubated purified recombinant GDF-8 and purified recombinantGDF-8 propeptide with conditioned media from COS1 cells transfected witheither a vector control or GASP1-V5-His. We then immunoprecipitatedGDF-8 with JA16-conjugated agarose beads and looked for co-purificationof GASP1 and GDF-8 propeptide using western blots (FIG. 15A). Both GASP1(lane 3) and GDF-8 propeptide (lane 1) co-immunoprecipitated with GDF-8,proving that GDF-8 can interact with both of these proteins. NeitherGASP1 nor propeptide were detected in JA16 immunoprecipitates in theabsence of GDF-8 (lane 4), eliminating the possibility of non-specificbinding in these experiments. When all three proteins were present, bothGASP1 and GDF-8 propeptide were pulled down with GDF-8, suggesting thepossibility that these proteins may form a tertiary complex (lane 5).However, this experiment does not eliminate the possibility that GASP1and propeptide are bound to the same epitope on separate GDF8 molecules.

To further confirm the interaction between GASP1 and GDF-8, we performedthe reverse immunoprecipitation by pulling down GASP1 from conditionedmedia supplemented with GDF-8 and/or GDF-8 propeptide recombinantprotein. To achieve this, we used an agarose-conjugated monoclonalantibody directed against the V5 epitope of the C-terminal V5-His tag onGASP1. As expected, GDF-8 co-immunoprecipitated with GASP1 (FIG. 15B,lanes 3 and 5), further confirming a direct interaction between theseproteins. Surprisingly, GDF-8 propeptide also co-purified with GASP1,even in the absence of GDF-8 (lane 4), suggesting that GDF-8 propeptidecan bind directly to GASP1. Thus, GASP1 binds to both GDF-8 and GDF-8propeptide independently. This is in contrast to FLRG, anotherfollistatin-domain protein, that binds exclusively to mature GDF-8 (Hillet al. (2002) J. Biol. Chem., 277: 40735-40741). Addition of both GDF-8and propeptide consistently showed less propeptide binding to GASP1 thanwhen propeptide was added alone. This observation suggests that GASP1may not bind to the GDF-8 small latent complex.

Example 11 GASP1-Mediated Inhibition of GDF-8 and BMP-11, But NotActivin or TGF-β1, Activity

A luciferase reporter construct, pGL3-(CAGA)₁₂ (SEQ ID NO:53) (Dennleret al. (1998) EMBO J., 17: 3091-3100) was transiently transfected intoA204 or RD rhabdomyosarcoma cells. Dilutions of conditioned media fromvector or GASP1 transfected cells were incubated for 30 minutes at 37°C. with 10 ng/ml GDF-8, 10 ng/ml BMP-11, 10 ng/ml rh activin A (R&DSystems), or 0.5 ng/ml rh TGF-β1 (R&D Systems). Luciferase activity wasmeasured according to Thies et al. (2001) Growth Factors, 18: 251-259and Zimmers et al. (2002) Science, 296: 1486-1488. In this assay, A204cells respond to GDF-8, BMP-11, and activin, but do not respond well toTGF-β1. RD cells respond to both GDF-8 and TGF-β1. Thus, we used A204cells to test for the ability of GASP1 to inhibit GDF-8, BMP-11, andactivin and RD cells to monitor the activity of TGF-β and GDF-8. Resultsfor GDF-8 are shown from A204 cells, but were similar in RD cells. Astandard curve measuring the concentration dependence of the luciferaseactivity induced by each of these growth factors was generated for eachexperiment (data not shown). The growth factor concentrations used fallin the linear region of this curve such that small changes inconcentration result in measurable changes in luciferase activity.

Two follistatin-domain proteins, follistatin and FLRG inhibit GDF-8activity in a (CAGA)₁₂ (SEQ ID NO:53) luciferase transcriptionalreporter assay, but also inhibit the biological activity of the relatedproteins, activin and BMP-11. The ability of GASP1 to inhibit GDF-8,BMP-11, activin, and TGF-β1 activity in the (CAGA)₁₂ (SEQ ID NO:53)reporter assay was also tested.

Various dilutions of conditioned media from COS cells transfected withV5-His tagged GASP1 or a vector control were incubated with purifiedrecombinant GDF-8 (10 ng/ml), BMP-11 (10 ng/ml), activin (10 ng/ml), orTGF-β1 (0.5 ng/ml) and assayed for growth factor activity inrhabdomyosarcoma cells expressing the (CAGA)₁₂ (SEQ ID NO:53) reporterconstruct. GASP1 potently inhibited GDF-8 activity in a concentrationdependent manner (FIG. 16A). GASP1 similarly inhibited the activity ofBMP-11 in this assay (FIG. 16B), as might be expected since mature GDF-8and BMP-11 are highly conserved and differ by only 11 amino acids.Surprisingly, GASP1 did not inhibit the activity of activin or TGF-β1(FIG. 16C and D), suggesting a very high level of specificity, which isnot demonstrated by follistatin itself. Thus, GASP1 exhibits specificityin its inhibition of GDF-8 and BMP-11.

The affinity of GASP1 for GDF-8 was evaluated by determining the IC50for inhibition of GDF-8 in the reporter gene assay. GASP1-V5-His proteinwas purified from conditioned media on a cobalt affinity column andeluted as described above. Fractions containing GASP1 were furtherpurified by size exclusion chromatography in PBS using a BioSepS3000column (Phenomenex). As shown in FIG. 17, GASP1 inhibited GDF-8 with anIC50 of approximately 3 nM.

Example 12 Treatment of Muscle Disorders

GASP1 may be administered to patients suffering from a disease ordisorder related to the functioning of GDF-8 according to Table 2.Patients take the composition one time or at intervals, such as oncedaily, and the symptoms of their disease or disorder improve. Forexample, symptoms related to a muscle disorder are improved, as measuredby muscle mass, muscle activity, and or muscle tone. This shows that thecomposition of the invention is useful for the treatment of diseases ordisorders related to the functioning of GDF-8, such as muscle disorders.

Table 2: Administration of GASP1

TABLE 2 Administration of GASP1 Route of Dosage Patient DiseaseAdministration Dosage Frequency Predicted Results 1 muscularsubcutaneous 25 mg once daily increase in muscle dystrophy mass andimprovement in muscle activity 2 muscular ″ 50 mg ″ increase in muscledystrophy mass and improvement in muscle activity 3 muscular ″ 50 mgonce weekly increase in muscle dystrophy mass and improvement in muscleactivity 4 muscular ″ 50 mg once monthly increase in muscle dystrophymass and improvement in muscle activity 5 muscular intramuscular 25 mgonce daily increase in muscle dystrophy mass and improvement in muscleactivity 6 ″ 50 mg ″ increase in muscle mass and improvement in muscleactivity 7 muscular ″ 50 mg once weekly increase in muscle dystrophymass and improvement in muscle activity 8 muscular ″ 50 mg once monthlyincrease in muscle dystrophy mass and improvement in muscle activity 9muscular intravenous 25 mg once daily increase in muscle dystrophy massand improvement in muscle activity 10 ″ 50 mg ″ increase in muscle massand improvement in muscle activity 11 muscular ″ 50 mg once weeklyincrease in muscle dystrophy mass and improvement in muscle activity 12muscular 50 mg once monthly increase in muscle dystrophy mass andimprovement in muscle activity 13 diabetes subcutaneous 50 mg once dailyimprovement in the management of blood sugar levels 14 ″ ″ 50 mg onceweekly improvement in the management of blood sugar levels 15 ″intramuscular 50 mg ″ improvement in the management of blood sugarlevels 16 ″ intravenous 50 mg ″ improvement in the management of bloodsugar levels 17 obesity subcutaneous 50 mg once daily weight loss andincrease in muscle mass 18 ″ intramuscular 50 mg once weekly weight lossand increase in muscle mass 19 intravenous 50 mg ″

The entire contents of all references, patents and published patentapplications cited throughout this application are herein incorporatedby reference. The foregoing detailed description has been given forillustration purposes only. A wide range of changes and modificationscan be made to the embodiments described above. It should therefore beunderstood that it is the following claims, including all equivalents,are intended to define the scope of the invention.

1. A method of treating a patient suffering from a muscle disorderassociated with GDF-8, comprising administering to the patient in needthereof a therapeutically effective dose of a protein comprising atleast one follistatin domain, wherein the protein is follistatin relatedgene (FLRG), and thereby improving a GDF-8 related biological outcome inthe patient, wherein the biological outcome is chosen from an increasein muscle mass, an increase in muscle strength, decreased adiposity, andimproved glucose homeostasis, thereby treating the patient sufferingfrom a muscle disorder.
 2. The method of claim 1, wherein the musculardisorder is muscular dystrophy.
 3. The method of claim 2, wherein themuscular dystrophy is chosen from severe or benign X-linked musculardystrophy, limb-girdle dystrophy, facioscapulohumeral dystrophy,myotonic dystrophy, distal muscular dystrophy, progressive dystrophicophthalmoplegia, oculopharyngeal dystrophy, and Fakuyama-type congenitalmuscular dystrophy.
 4. A method of inhibiting or reducing a GDF-8activity to achieve a desired biological outcome, comDrisingadministering to a mammal at least one protein comprising at least onefollistatin domain, wherein the protein is FLRG, and wherein the proteininhibits a GDF-8 activity, thereby inhibiting or reducing the GDF-8activity and improving a GDF-8 related biological outcome in the mammal,wherein the biological outcome is chosen from an increase in musclemass, an increase in muscle strength, decreased adiposity, and improvedalucose homeostasis.